Studies of elementary techniques for developing on-chip devices for single genomic DNA manipulation and analysis

通过这项研究，我们发展了一种新的方法来观察和人工控制基因组DNA的高阶结构，该方法能够以较高的重复性对超过250μm长的DNA进行可逆折叠和去折叠。在真核细胞中，DNA在组蛋白等阳离子蛋白的帮助下折叠形成高度有序的染色体结构，基因表达受到特定基因折叠紧密程度的影响，这是由组蛋白甲基化或乙酰化等过程控制的。然而，DNA折叠的机制和动力学，如DNA链上的折叠速度，或协同作用(折叠开始后折叠速度会增强)对超长DNA链的折叠有多大影响，目前还不清楚。这样的研究需要一种方法来拉伸DNA链，将其保存在溶液中，该溶液可以迅速取代不同阳离子或盐浓度的溶液。为此，我们研制了一种微流控装置。我们研制的器件是用PDMS进行标准软光刻的。该装置的主通道有微柱，通过流体动力将DNA钩住并拉伸。在柱子的上游一侧有入口，酵母染色体DNA、1 mM亚精胺(DNA缩合聚阳离子)和500 mM氯化钠(去缩合盐)从这里喂养。每种溶液含有1个μM YO-PRO-1，用于DNA的荧光可视化。该方法不仅有助于基础的生化研究，而且可能为DNA处理向单分子测序或通过人工诱导的高阶改变来控制细胞活动开辟道路。

Through this research, we developed a new methodology for the observation and the artificial control of higher-order structures of genomic DNA molecules, and the method enables reversible folding and unfolding of over 250 μm-long DNA with a high reproducibility.In eukaryotic cells, DNA with the aid of cationic proteins such as histone are folded to form a highly ordered chromosome structures, and the gene expression is influenced by how tightly it is fold around a particular gene, which is controlled by such a process as histone methylation or acetylation. However, the mechanism and the dynamics of DNA folding, such as the speed of folding on a DNA strand, or how much cooperativity (the folding speed is enhanced once folding starts) influences the folding of very long DNA strand, is not well understood.Such a study requires a method to stretch DNA strands, hold it in a solution, which can be quickly replaced the solutions having different cationic or salt concentrations. For this purpose, we have developed a microfluidic device. The device which we developed is fabricated by a standard soft lithography with PDMS. The main channel of the device has micro-pillars to hook and stretch DNA by hydrodynamic force. There are inlets on the upstream side of the pillars, from which yeast chromosomal DNA, 1 mM spermidine (DNA condensing polycationic agent), and 500 mM NaCl (de-condensing salt) are fed. Each solution contains 1 μM YO-PRO-1 for fluorescent visualization of DNA.The method developed here not only contributes to the basic biochemical researches, but may also open a way for the DNA handling towards single-molecule sequencing, or control of cellular activity through artificially induced higher-order changes.